Cloning, Organization, and Expression of the Bioluminescence Genes ...

Vol. 172, No. 10

JOURNAL OF BACTERIOLOGY, Oct. 1990, p. 5767-5773 0021-9193/90/105767-07$02.00/0 Copyright C) 1990, American Society for Microbiology

Cloning, Organization, and Expression of the Bioluminescence Genes of Xenorhabdus luminescens SUSAN FRACKMAN,* MICHAEL ANHALT, AND K. H. NEALSON

Center for Great Lakes Studies, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53204 Received 11 May 1990/Accepted 20 July 1990

The lux genes of Xenorhabdus luminescens, a symbiont of the nematode Heterorhabditis bacteriophora, were cloned and expressed in Escherichia coli. The expression of these genes in E. coli was qualitatively similar to their expression in X. luminescens. The organization of the genes is similar to that found in the marine luminous bacteria. Hybridization studies with the DNA that codes for the two subunits of luciferase revealed considerable homology among all of the strains of X. luminescens and with the DNA of other species of luminous bacteria, but none with the nonluminous Xenorhabdus species. Gross DNA alterations such as insertions, deletions, or inversions do not appear to be involved in the generation of dim variants known as secondary forms.

Xenorhabdus luminescens is a luminous bacterium in the family Enterobacteriaceae (3, 17, 38). X. luminescens strains are symbiotic with the entomopathogenic nematodes Heterorhabditis bacteriophora (2, 16, 32). The bacteria are carried in specialized intestinal vesicles (4) of the nonfeeding stage of the nematode. When the nematode locates a suitable insect, it penetrates into the hemocoel and releases its bacterial symbionts. Within the hemocoel, the bacteria grow and are involved in the killing of the insect host. The bacteria create an optimal environment for nematode reproduction and serve as a source of food for the nematode. Upon nutrient depletion, the nematodes molt into third-instar nonfeeding larvae (32), take on X. luminescens symbionts, and emerge from the insect cadaver in search of new prey (16, 32). Besides bioluminescence, X. luminescens produces several other interesting products: antibiotic substances (2, 30, 33), extracellular proteases (3, 35), extracellular lipase (3, 17), intracellular protein crystals (10), and a red pigment (33). The insect cadaver does not putrefy, presumably because the antibiotic activity inhibits the growth of other bacteria. The extracellular protease and lipase may function to break down proteins and lipids of the insect carcass, thereby providing nutrients for bacterial and nematode growth. Bioluminescence and the red pigment are postulated to have a role in dissemination by attracting potential prey insects. The function of the intracellular protein crystals has not been determined. X. luminescens exhibits two distinct forms called primary and secondary forms (1, 6, 7, 18). The primary form is generally isolated from infective nematodes, but upon prolonged culture in various media secondary variants appear. In contrast to the primary form, the secondary variants are dim and lack detectable protease, lipase, antibiotic activity, protein crystals, and red pigment (6). The two forms also exhibit differences in colony morphology and staining properties (18). The secondary forms are deficient in providing optimum conditions for nematode reproduction (1, 7, 16). The physical and biochemical properties of the luminous system of X. luminescens are similar to those found in other bioluminescent bacteria (9, 31, 36). The enzyme luciferase, an alpha-beta heterodimeric mixed-function oxidase, catalyzes the oxidation of reduced flavin and long-chain alde*

hyde to oxidized flavin and the corresponding long-chain fatty acid. A fatty acid reductase complex is required for the generation and recycling of fatty acid to aldehyde (34), and an NAD(P)H:flavin oxidoreductase supplies the reduced flavin (20). In rich media, the luminescence of X. luminescens increases dramatically during the late logarithmic or stationary phase of growth (36). The generation of secondary variants which are dim is another level of control of the luminous system of X. luminescens. The bioluminescence (lux) genes from Vibrio harveyi (8), Vibrio fischeri (12), Photobacterium leiognathi (11), and Photobacterium phosphoreum (22) have been cloned and expressed in Escherichia coli. Five structural genes are required for light emission: luxC, luxD, and luxE encode the fatty acid reductase complex, and luxA and luxB encode the alpha and beta subunits of bacterial luciferase (12, 13, 25). In all luminous bacteria that have been studied, these five genes are closely linked and luxA and luxB are flanked by the genes for the enzymes of the fatty acid reductase complex. In both P. phosphoreum and P. leiognathi there is another gene between luxB and luxE (19, 22). The function of the product of this gene is not known. In this paper we report the cloning of the lux genes of X. luminescens and their expression when introduced into E. coli. We also present data showing the organization of the X. luminescens lux genes, the structure of the lux genes from the secondary form, and the homology of the luxA and luxB genes of X. luminescens Hm to DNA of other X. luminescens strains and other bioluminescent bacteria. MATERIALS AND METHODS

Bacterial strains and media. The bacterial strains used in this study are presented in Table 1. Cells were grown in L broth, consisting of 1% tryptone (Difco Laboratories), 0.5% yeast extract, and 1% NaCl (26). For solid medium, 1.5% Bacto-Agar (Difco) was added. L broth and L agar were supplemented with ampicillin (100 pLg/ml), 5-bromo-4-chloro5-indolyl-p-D-galctopyranoside (X-Gal; 40 ,ug/ml), or isopropyl-3-D-thiogalactopyranoside (IPTG; 0.4 mM) as needed. Growth conditions and luminescence measurements. Growth experiments were performed with 50 ml of media in a 250-ml flask at 28 or 30°C on a rotary shaker. At timed intervals, samples were removed for measurement of optical density (OD) and luminescence. The OD at 560 nm (OD560) was measured on an LKB Ultraspec 4050 spectrophotometer,

Corresponding author. 5767

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TABLE 1. Bacterial strains used in this work

Relevant phenotype or genotype

Strain

X. luminescens Hm Hm 22A Fla NC19 NC19 X. poinarii

Source or reference

Primary form Secondary form Primary form Primary form Primary form Secondary form Primary form (Lux-)

6 6 J. Ensign J. Ensign 3 This work 3

RESULTS

E. coli LE392 DH5otF,lq

27 F' lacIq lacZAM15 Bethesda Research Laboratories TnS(KmD)

Other luminous bacteria: P. leiognathi PL721 P. phosphoreum NZ11D V. harveyi B392 V. fischeri MJ1 V. cholerae (V. albensis) ATCC 14547

V. vulnificus 21

DNA probes were labeled with [a-32P]dATP (Du Pont, NEN Research Products) by the random primer method (14) or by nick translation (23). Southern blot hybridizations (37) were performed by standard methods (23). For high-stringency analysis hybridizations were carried out in 50% formamide at 42°C and washes were carried out in 0.1x SSC (lx SSC is 0.15 M NaCl plus 0.015 sodium citrate) at 42°C. For low-stringency analysis, hybridizations were carried out in 50% formamide at room temperature and washes were carried out in 2x SSC at room temperature.

Lux' Lux'

Out collection Our collection

Lux' Lux' Lux'

Our collection Our collection Our collection

Lux'

J. Oliver

and luminescence was measured on a photomultiplier photometer as previously described (28). Recombinant DNA procedures. Enzymes and reagents were purchased from Bethesda Research Laboratories, Inc., and Promega Biotec. Standard procedures were used for cloning, subcloning, and restriction mapping (23). Some deletions were generated by linearizing plasmid pCGLS1 with either restriction endonuclease SstI or PstI and then treating it with exonuclease Bal3l (23). Genomic DNA was isolated by a modification of the procedure describe by Fulton et al. (15). Plasmid DNA was purified by the method of Bimboim and Doly (5). E. coli cells were made competent for transformation by the method of Kushner (21).

Cloning of the bioluminescence genes. A genomic library of X. luminescens Hm DNA was prepared by partial digestion with Sau3A, enrichment for fragments of 9 to 20 kilobases (kb) by agarose gel electrophoresis, ligation of these fragments into the BamHI site of plasmid pUC18, and transformation of these plasmids into E. coli. The transformants were screened for light production. One luminous colony was identified which carried a plasmid, pCGLS1, with an insert of approximately 11 kb of X. luminescens DNA. A partial restriction map of this plasmid is shown in Fig. 1. It is known from the cloning of other luminous systems that the production of light without the addition of exogenous aldehyde in E. coli requires the expression of at least five genes, luxA through luxE (12, 13, 25). These genes encode the two subunits of luciferase and the enzymes of the fatty acid reductase complex. Therefore, by analogy, it appeared that pCGLS1 carried at least the genes for luciferase and the fatty acid reductase complex, and they were designated lux to maintain the nomenclature adopted for the other bacterial bioluminescence systems. Expression of the lux genes in E. coli. When X. luminescens was grown in a rich medium such as L broth, its bioluminescence remained low until the cells reached the late logarithmic or early stationary phase of growth. The bioluminescence increased after the culture reached an OD560 of 2.0 or more; maximal light production occurred after the culture was in the stationary phase (OD560 > 4.0) (Fig. 2A

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FIG. 1. Map of the lux genes. The locations of the lux genes and the direction of transcription (arrow) are shown above the restriction map. The probes used in hybridization experiments are shown below the restriction map. Restriction endonuclease sites are abbreviated as follows: Bs, BstEII; C, ClaI; E, EcoRI; H, HindIII; M, MluI; S, ScaI, X, XbaI. B/Sa represents the joining of BamHI- and Sau3A-cut DNA.

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GROWTH (OD560) FIG. 2. Expression of the lux genes of X. luminescens. (A) Growth of X. luminescens expressed as cell density (OD560) as a function of time; (B) light production in X. luminescens expressed as light units normalized to cell density (OD560) as a function of cell density (OD56). (C) Growth and (D) light production of E. coli DH5aF,lq carrying pCGLS1 (@) or pCGLSlR (0).

and B). The production of light from E. coli DHaF'lq carrying pCGLS1 during growth (Fig. 2C and D) showed a similar pattern of development to that from X. luminescens. Two differences in light production by the E. coli strain carrying pCGLSl and X. luminescens were noted. The strain carrying pCGLS1 produced 10- to 15-fold more light than X. luminescens, and the magnitude of the increase in light production late in growth was approximately 10-fold smaller in the strain carrying pCGLS1 than in X. luminescens. These differences may be due to a gene dosage effect, since pCGLS1 is present in many copies per cell. The plasmid pCGLSlR was constructed to investigate whether the X. luminescens lux promoter(s) was present on the cloned fragment and functional in E. coli. This plasmid contains the same DNA insert as pCGLS1, but in the opposite orientation relative to the vector. The emission of light during growth was measured from E. coli DH5oF,lq carrying pCGLS1 and pCGLS1R. This host strain carries the lacIq repressor allele on an F' episome which overproduces the Lac repressor protein; therefore, the lac promoter that is present on the vector should be repressed in these studies. The emission of light during growth from cells carrying pCGLS1 and pCGLSlR was virtually identical (Fig. 2C and D), suggesting that the lux genes were expressed from a

promoter(s) within the X. luminescens DNA insert rather than from promoters on the vector. Organization of the lux genes. Expression of the genes which encode luciferase and the enzymes of the fatty acid reductase complex is required for light production without the addition of exogenous aldehyde in E. coli, whereas only the genes for luciferase are required for light emission if long-chain aldehyde is supplied. The organization of the cloned lux genes of X. luminescens was determined by analyzing the ability of plasmids carrying deletions in the lux region to produce aldehyde-independent and -dependent luminescence. A series of deletions of the lux insert of pCGLS1 was constructed (Fig. 3). The smallest DNA region that allowed aldehyde-independent luminescence was the 6.9-kb insert of pCGLS11. The amount of light emitted from E. coli carrying a plasmid with this insert is different depending upon the orientation of the insert with respect to the plasmid sequences (data not shown), suggesting that the expression of the lux genes is under the control of the plasmid promoter(s). It is therefore possible that part or all of the natural X. luminescens promoter for the lux genes was deleted in pCGLS11. Plasmids with deletions from either the left end (pCGLS5, pCGLS7, or pCGLS15) or the right end

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INSERT BGI 'Sa pCGLS I

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FIG. 3. Deletion analysis of the lux genes. Lines indicate the regions of X. luminescens DNA that have been inserted into pUC18 or pUC19 and transformed into E. coli LE392. The restriction endonuclease abbreviations are defined in the legend to Fig. 1. The ability (+) or inability (-) to produce light either in the absence (-aldehyde) or after the addition (+aldehyde) of 0.1% decanal (in dimethyl sufloxide to a final concentration of 0.002%) is indicated for each plasmid.

(pCGLS20) of the lux DNA insert produced aldehyde-dependent luminescence, indicating that the genes that encode luciferase (luxA and luxB) are located in the 3.1-kb region between the left end of pCGLS15 and the right end of pCGLS20 and that the genes for aldehyde biosynthesis are present both to the right and the left of luxA and luxB (Fig. 1). Direction of transcription of the lux genes. Plasmids were constructed which carry the E. coli lac promoter either to the left or to the right of the cloned lux genes (Fig. 4). The lac promoter is oriented so that it directs transcription through the inserted lux region. When the lac promoter is oriented in the same direction as the natural lux promoter, there will be an increase in light emission upon induction of the lac promoter; no increase is expected when the induced lac promoter is in the opposite orientation. The results of these experiments are shown in Fig. 4. Plasmids pCGLS11 and pCGLS5 have the lac promoter to the left of the lux genes, and strains carrying these plasmids produced 6.7- and 12.0fold more light, respectively, in response to lac induction. In contrast, pCGLS11R and pCGLS5R have the lac promoter to the right of the lux genes, and no enhancement of luminescence was seen upon lac induction. These results indicate that the lux genes are transcribed from left to right. Strains carrying pCGLS1 and pCGLSlR provided an interesting result, since both orientations displayed enhanced luminescence in response to lac induction (15- and 7.3-fold, respectively). These results suggest that there may be transcription in both directions from the lux DNA region. Rightward transcription produces luciferase and the enzymes of the fatty acid reductase complex (Fig. 1), whereas leftward transcription may produce a product that positively affects the expression of the lux genes. The existence of a

leftward transcript is very speculative and requires further investigation. Relationship of X. luminescens lux genes to those of other luminous bacteria. The homology between luxA and luxB of X. luminescens Hm and the DNA of other X. luminescens strains was investigated by Southern hybridization analysis. DNA samples from various Xenorhabdus strains were hybridized at high stringency with a probe carrying most of luxA and part of luxB (Fig. 1, probe C). The results indicated that all strains of X. luminescens tested (Hm, 22A, Fla, and NC19) contained DNA that hybridized with the probe from strain Hm (Fig. 5). All the strains showed strong hybridization to the probe. DNA from secondary variants of Hm and NC19 were included in this analysis. The lux genes were present in the secondary variants with no apparent rearrangements or alterations in copy number. There was no hybridization of the lux probe to DNA from the nonluminous species Xenorhabdus poinarii. The relationship of the luxA and luxB genes of X. luminescens to those of other genera of bioluminescent bacteria was also determined by Southern hybridization analysis. Under low-stringency conditions, a probe carrying the X. luminescens luxA gene and a part of luxB (Fig. 1, probe D) hybridized to all of the bioluminescent bacteria that were tested (Fig. 6), with the Photobacterium species DNA showing weak hybridization and the Vibrio species DNA showing strong hybridization. Structure of the lux genes in secondary variant. One possible explanation for the difference in bioluminescence between the primary and secondary variants is that there is a chromosomal rearrangement in the secondary variant, resulting in reduced expression. To investigate this possibility, we compared the structures of the lux DNA regions of

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+IPTG/ -IPTG I

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FIG. 4. Enhancement of lux expression following induction of the vector-encoded lac promoter. Lines indicate the regions of X. luminescens DNA that have been inserted into pUC18 or pUC19. The arrows above or below the lines indicate the direction of transcription from the E. coli lac promoter that is present on the vector. The ratio of luminescence under conditions in which the lac promoter is induced (+IPTG) to luminescence under conditions in which the lac promoter remains uninduced (-IPTG) is given for each plasmid. The plasmids were in E. coli DH5aF'lq. Light was measured when the cultures were at an OD560 of approximately 2.0, and each number represents the average of results from three independent samples. Restriction endonuclease abbreviations are defined in the legend to Fig. 1. For cells carrying pCGLS5 and pCGLS5R, light was measured following the addition of decanal as described in the legend to Fig. 3.

primary and secondary forms of X. luminescens Hm by Southern hybridization analysis. DNA samples from the primary and secondary forms were digested with various restriction endonucleases. In all, 16 enzymes (AluI, ClaI, DraI, EcoRI, HhaI, HincII, HindIII, Hinfl, MnlI, MseI, RsaI, Sau3A, ScrFI, TaqI, and ThaI) were used for the analysis. Three different hybridization probes (Fig. 1, probes

1

2

3 4 5 6

7

-12 wW_

A, B, and C), which spanned the entire 11-kb insert of pCGLS1, were used. In no case was any difference seen between the DNA structure of the primary and secondary forms (data not shown). DISCUSSION Organization of the lux genes. In all bioluminescent bacteria that have been studied, the genes encoding the two subunits of luciferase and the enzymes of the fatty acid reductase complex are closely linked and organized in a similar fashion (25). The data presented here indicate that the organization of the lux genes of X. luminescens is similar 2 3 4 5 6 7 8 9 10

1 I

-9

-8

7 -6 -

as

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FIG. 5. Southern blot analysis of the lux genes of X. luminescens strains. Genomic DNA was prepared from X. luminescens strains, cut with restriction endonuclease EcoRI, electrophoresed on a 1% agarose gel, blotted, and hybridized to a probe prepared from pCGLS1 (Fig. 1, probe C). Lanes: 1, primary isolate of X. luminescens Hm; 2, secondary isolate of X. luminescens Hm; 3, primary isolate of X. luminescens 22A; 4, primary isolate of X. luminescens Fla; 5, primary isolate of X. luminescens NC19; 6, secondary isolate of X. luminescens NC19; 7, primary isolate of X. poinarii.

- 8^

40

4.0

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1.0

FIG. 6. Southern blot analysis of the lux genes of bioluminescent bacteria. Genomic DNA was prepared from strains of bioluminescent bacteria, cut with restriction endonuclease EcoRI, electrophoresed on a 1% agarose gel, blotted, and hybridized to a probe prepared from pCGLS1 (Fig. 1, probe D). Lanes: 1, primary isolate of X. luminescens; 2, secondary isolate of X. luminescens; 3, P. leiognathi; 4, P. phosphoreum; 5, V. harveyi; 6, V. fischeri; 7, V. vulnificus; 8, V. cholerae; 9, X. poinarii; 10, E. coli.

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FRACKMAN ET AL.

to that in the other luminous bacteria, with the genes for the two subunits of luciferase flanked by the genes for the enzymes of the fatty acid reductase complex. In contrast to the structural genes of the various luminous bacteria, the genes which regulate lux gene expression are not conserved in their genetic organization. In V. fischeri, two regulatory genes that are closely linked to the structural genes have been identified. The luxI gene, which encodes a protein necessary for autoinduction, is in the same operon as luxA through luxE, and the luxR gene, which encodes a positive regulatory protein, is located in an operon adjacent to the lux structural genes (12, 13). In V. harveyi an unlinked regulatory gene has been identified (24). The regulatory genes of the X. luminescens system have not been identified. However, the cloned DNA fragment regulates the synthesis of the luminous system in a manner similar to that seen in the wild type, suggesting either that the genes that regulate the luminous systems of X. luminescens are present in E. coli or that regulatory genes are present on the 11-kb pCGLS1 DNA insert. Our studies on the direction of lux transcription provide evidence for the presence of a closely linked transcriptional unit which encodes a positive regulatory gene. Relationship of luxA and luxB from X. luminescens to the genes encoding luciferase from other luminous bacteria. The DNA hybridization experiments indicated that there is a strong homology to the luxA and luxB genes of X. luminescens Hm in all of the strains of X. luminescens including secondary variants. In contrast, the nonlimunous species, X. poinarii, showed no hybridization with the luxAB probe even at low stringency. This type of hybridization analysis may be a valuable approach to the identification of naturally occurring secondary variants and thereby provide a means to investigate the ecological significance of the formation of secondary variants. The luxA and luxB probe of X. luminescens showed some homology to DNA isolated from all of the other luminous bacteria that were tested. There was strong hybridization of the X. luminescens probe to the DNA from the Vibrio species, whereas much less hybridization was observed to the DNA of the Photobacterium species. These data are consistent with the results of experiments in which isolated subunits of X. luminescens luciferase formed active hybrids with subunits of V. harveyi and V. cholerae luciferase (36). The conservation of sequence, function, and genetic organization strongly suggests that the luminous systems of all of these bacteria have a common origin. Secondary variants. A trait common to most luminous bacteria is the formation of spontaneous dim or dark variants (29). This is seen in X. luminescens with the formation of secondary variants (1, 6, 7, 18). These secondary variants are deficient in the production of several other bacterial products and also have deceased ability to support the life cycle of their nematode hosts. The mechanism of conversion to dim or dark variants is unknown for all of the luminous species. On the basis of extensive analysis of endonuclease restriction sites in both the primary and secondary form, it appears that the structure of the lux genes of the secondary form of X. luminescens is not different from the structure of the lux genes of the primary form. This strongly suggests that the conversion from primary to secondary form is not mediated by a DNA deletion, insertion, or inversion within the lux genes. This does not rule out the possibility of a very small DNA alteration within the lux genes. The lux gene sequences of primary and secondary forms will have to be compared before this possibility can be ruled out. Other

J. BACTERIOL.

mechanisms of conversion from the primary to the secondary form are currently under investigation in our laboratory. ACKNOWLEDGMENTS We thank David Bowen, Jerry Ensign, Woody Hastings, and J. Oliver for strains. We also thank Barbara Wimpee for artwork and Loraine Samsel for manuscript preparation. This work was supported by U.S. Department of Agriculture grant 8800776.

LITERATURE CITED 1. Akhurst, R. J. 1980. Morphological and functional dimorphism in Xenorhabdus spp., bacteria symbiotically associated with the insect pathogenic nematodes Neoaplectana and Heterorhabditis. J. Gen. Microbiol. 121:303-309. 2. Akhurst, R. J. 1982. Antibiotic activity of Xenorhabdus spp., bacteria symbiotically associated with insect pathogenic nematodes of the families Heterorhabditidae and Steinernematidae.

J. Gen. Microbiol. 128:3061-3065. 3. Akhurst, R. J., and N. E. Boemare. 1988. A numerical taxonomic study of the genus Xenorhabdus (Enterobacteriaceae) and proposed elevation of the subspecies of X. nematophilus to species. J. Gen. Microbiol. 134:1835-1845. 4. Bird, A. F., and R. J. Akhurst. 1983. The nature of the intestinal vesicle in nematodes of the family Steinernematidae. Int. J. Parasitol. 13:599-606. 5. Birnboim, H., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 6. Bleakley, B., and K. H. Nealson. 1988. Characterization of primary and secondary forms of Xenorhabdus luminescens strain Hm. FEMS Microbiol. Ecol. 53:241-250. 7. Boemare, N., and R. J. Akhurst. 1988. Biochemical and physiological characterization of colony form variants in Xenorhabdus spp. (Enterobacteriaceae). J. Gen. Microbiol. 134:751-761. 8. Cohn, D. H., R. Ogden, J. Abelson, T. Baldwin, K. Nealson, M. Simon, and A. J. Mileham. 1983. Cloning of the Vibrio harveyi luciferase genes: use of a synthetic oligonucleotide probe. Proc. Natl. Acad. Sci. USA 80:120-123. 9. Colepicolo, P., K. Cho, G. 0. Poinar, and J. W. Hasting. 1989. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl. Environ. Microbiol. 55: 2601-2606. 10. Couche, G., and R. Gregson. 1987. Protein inclusions produced by the entomopathogenic bacterium Xenorhabdus nematophilus subsp. nematophilus. J. Bacteriol. 169:5279-5288. 11. Delong, E. F., D. Steinhauer, A. Israel, and K. Nealson. 1987. Isolation of the lux genes from Photobacterium leiognathi and their expression in Escherichia coli. Gene 54:203-210. 12. Engebrecht, J., K. Nealson, and M. Silverman. 1983. Bacterial bioluminescence: isolation and genetic analysis of functions from Vibriofischeri. Cell 32:773-781. 13. Engebrecht, J., and M. Silverman. 1984. Identification of genes and gene products necessary for bacterial bioluminescence. Proc. Natl. Acad. Sci. USA 81:4154-4158. 14. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13. 15. Fulton, G. L., D. N. Nunn, and M. E. Lidstrom. 1984. Molecular cloning of a malyl coenzyme A lyase gene from Pseudomonas sp. strain AM1, a facultative methylotroph. J. Bacteriol. 160: 718-723. 16. Gaugler, R. 1988. Ecological considerations in the biological control of soil-inhabiting insects with entomopathogenic nematodes. Agric. Ecosyst. Environ. 24:351-360. 17. Grimont, P., A. Steigerwalt, N. Boemare, F. Hickman-Brenner, C. Deval, F. Grimont, and D. Brenner. 1984. Deoxyribonucleic acid relatedness and phenotypic study of the genus Xenorhabdus. Int. J. Syst. Bacteriol. 34:378-388. 18. Huribert, R. E., J. Xu, and C. Small. 1989. Colonial and cellular polymorphism in Xenorhabdus luminescens. Appl. Environ. Microbiol. 55:1136-1143.

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19. Illarionov, B., M. Protopopova, V. Karginov, N. Mertvetsov, and J. Gitelson. 1988. Nucleotide sequence of part of Photobacterium leiognathi lux region. Nucleic Acids Res. 16:9855. 20. Jablonski, E., and M. DeLuca. 1978. Studies of the control of luminescence in Beneckea harveyi: properties of the NADH and NADH:FMN oxidoreductases. Biochemistry 17:672-678. 21. Kushner, S. R. 1978. Improved method for the transformation of E. coli with colEl derived plasmids, p. 17-21. In H. Boyer and S. Nicosia (ed.), Genetic engineering. Proceedings of the International Symposium on Genetic Engineering. Elsevier/North Holland Publishing Co., Amsterdam. 22. Mancini, J., M. Boylan, Rk. Soly, A. Graham, and E. Meighen. 1988. Cloning and expression of the Photobacterium phosphoreum luminescence system demonstrates a unique lux gene organization. J. Biol. Chem. 263:14308-14314. 23. Maniatis, T., E. F. Fritsclh, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 24. Martin, M., R. Showalter, and M. Silverman. 1989. Identification of a locus controlling expression of luminescence genes in Vibrio harveyi. J. Bacteriol. 171:2406-2414. 25. Meighen, E. A. 1988. Enzymes and genes from the lux operons of bioluminescent bacteria. Annu. Rev. Microbiol. 42:151-176. 26. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 27. Murray, N. E., W. J. Brammer, and K. Murray. 1977. Lambdoid phages that simplify the recovery of in vitro recombinants. Mol. Gen. Genet. 150:53-58. 28. Nealson, K. H. 1978. Isolation, identification and manipulation of luminous bacteria. Methods Enzymol. 57:153-166. 29. Nealson, K. H., and J. W. Hastings. 1979. Bacterial biolumines-

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cence: its control and ecological significance. Microbiol. Rev. 43:496-518. Paul, V. J., S. Frautschy, W. Fenical, and K. Nealson. 1981. Antibiotics in microbial ecology: isolation and structure assignment of several new antibacterial compounds from the insectsymbiotic bacteria Xenorhabdus spp. J. Chem. Ecol. 7:589-597. Poinar, G., G. Thomas, M. Haygood, and K. Nealson. 1980. Growth and luminescence of the symbiotic bacteria associated with the terrestrial nematode, Heterorhabditis bacteriophora. Soil Biol. Biochem. 12:5-10. Poinar, G. O., Jr. 1983. The natural history of nematodes. Prentice-Hall, Inc., Englewood Cliffs, N.J. Richardson, W., T. Schmidt, and K. Nealson. 1988. Identification of an anthraquinone pigment and a hydroxystilbene antibiotic from Xenorhabdus luminescens. Appl. Environ. Microbiol. 54:1602-1605. Riendeau, D., and E. Meighen. 1989. Co-induction of fatty acid reductase and luciferase during development of bacterial bioluminescence. J. Biol. Chem. 255:12060-12065. Schmidt, T., B. Bleakley, and K. Nealson. 1988. Characterization of an extracellular protease from the insect pathogen Xenorhabdus luminescens. Appl. Environ. Microbiol. 54:2793-2797. Schmidt, T., K. Kopecky, and K. Nealson. 1989. Bioluminescence of the insect pathogen Xenorhabdus luminescens. Appl. Environ. Microbiol. 55:2607-2612. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. Thomas, G., and G. Poinar. 1979. Xenorhabdus gen. nov., a genus of entomopathogenic nematophilic bacteria of the family Enterobacteriaceae. Int. J. Syst. Bacteriol. 29:352-360.